This proposal seeks to understand the mechanisms by which the productive life cycle of human papillomaviruses (HPV) is regulated using an in vitro raft culture system. It also seeks to develop methods for the testing of anti-papillomaviral agents. The life cycle of human papillomaviruses is closely linked to epithelial differentiation and in the past, studies on HPVs have been limited due to an inability to duplicate this program in vitro. Recently, an in vitro system (rafts) has been used which allows for the stratification and differentiation of keratinocytes at the air-liquid interface of collagen raft cultures to examine the life cycle of human papillomaviruses. Using raft cultures of cell lines (designated CIN-612) which stably maintain episomal copies of HPV, the productive life cycle of HPV type 3lb has been duplicated. In situ hybridization analysis of CIN612 raft cross sections demonstrated specific amplification of HPV3lb DNA in the upper layers similar to that seen in biopsies of low grade lesions. By the addition of phorbol esters to induce a more complete program of differentiation, the synthesis of capsid proteins was also induced. Virions of approximately 50 nm in size were detected by electron microscopes in suprabasal nuclei of TPA treated rafts. No such structures were seen in the nuclei of untreated cells. Using this system it has also been possible to identify a series of HPV3lb transcripts which were expressed constitutively throughout the stratified epithelium as well as a set of transcripts that are induced upon differentiation. This later class include transcripts for E1-E4 as well as for the late capsid genes L1 and L2. In this application, we propose to use the raft system for viral production to examine in detail the differentiation dependent life cycle of human papillomaviruses. In vitro assays will be developed with which anti-papillomaviral agents can be screened for their ability to inhibit replication. Specifically, we propose to: l). Characterize in detail the patterns of HPV3lb transcription by in situ analysis in raft cultures and examine distribution of viral proteins by immunohistochemistry and immunoprecipitation. 2). Define the cis and trans requirements for viral replication in basal cells as well as to define the mechanism by which amplification of genomes occurs in suprabasal cells. 3). Determine if transfected viral sequences from types 11 and 18 can remain episomal in keratinocyte cell lines following electroporation. Determine if these cell lines can then be induced to amplify their viral DNA and synthesize virions in raft cultures. 4). Examine the mechanism of induction of late L1/L2 expression by TPA treatment, posttranscriptional and translational control. 5). Develop methods by which to screen drugs for the ability to inhibit the HPV life cycle in vitro.